The field of the present invention relates to techniques for screening differences in gene expression between various cell types or between different stages of cell development.
In higher organisms, every cell expresses about 10-20% of the 100,000 possible different genes. Gene expression is involved in all life processes, such as development, aging and disease states. Thus, the analysis of which genes are expressed at any given time, and the identification of the expressed mRNAs, is of prime interest in molecular biology.
One such method for screening differences in gene expression is known as Differential Display. This method is described in Pardee et al., U.S. Pat. No. 5,262,311, hereby incorporated by reference. Differential Display involves amplifying partial cDNA sequences from subsets of mRNAs by reverse transcription and the polymerase chain reaction (PCR), then displaying these sequences on a sequencing gel.
In the Differential Display method, the primers which hybridize to the 3' end of the mRNA [the 3' primers] are selected to take advantage of the polyadenylate [polyA] tail present on most eukaryotic mRNAs to anchor the primers at the 3' end of the mRNA. Each 3' primer hybridizes to a portion of the polyA tail and additionally to 2 bases which are immediately 5' of the polyA tail. The 2 nucleotides of the 3' primer which are not complementary to the polyA tail are of the sequence VN, where V is deoxyadenylate ("dA"), deoxyguanylate ("dG"), or deoxycytidylate ("dC"), and N, the 3' terminal nucleotide, is dA, dG, dC, or deoxythymidylate ("dT"). By probability, each 3' primer Will recognize one-twelfth of the total mRNA population, since there are twelve different combinations of the two 3' bases, eliminating T as the base which hybridizes immediately 5' of the polyA tail. Such primers are used to reverse transcribe specific subpopulations of mRNAs.
A second set of primers, the 5' primers, is designed to randomly select a subset of the cDNAs generated using the 3' oligonucleotide primers. The 5' primers are of arbitrary base sequence. The cDNA sequences defined by these two primer sets are then amplified by PCR. The amplified products can then be displayed on a sequencing gel, and visualized by autoradiography. Using this method, comparisons can be made of the genes expressed by different cell types, or by cells in various stages of development or disease.
Differential Display uses short 9 to 14 base primers that require low temperature annealing throughout PCR amplification. Such low temperature annealing conditions result in a decline in the reproducibility of the method. As noted by the creators of Differential Display (Liang et al., Nucl. Acids Res. 21:3269-3275), "[a] troublesome aspect of the method is that the noise level of false positives, though a few percent, can be very appreciable relative to the truly different bands between cells."